title: Development of assays using hexokinase and phosphoglucomutase gene sequences that distinguish strains of Leishmania tropica from different zymodemes and microsatellite clusters and their application to Palestinian foci of cutaneous leishmaniasis.
abstract: OBJECTIVE
  Palestinian strains of L.tropica characterized by multilocus enzyme electrophoresis (MLEE) fall into two zymodemes, either MON-137 or MON-307.
  
  
  RESULTS
  Assays employing PCR and subsequent RFLP were applied to sequences found in the Hexokinase (HK) gene, an enzyme that is not used in MLEE, and the Phosphoglucomutase (PGM) gene, an enzyme that is used for MLEE, to see if they would facilitate consigning local strains of L.tropica to either zymodeme MON-137 or zymodeme MON-307. Following amplification and subsequent double digestion with the restriction endonucleases MboI and HaeIII, variation in the restriction patterns of the sequence from the HK gene distinguished strains of L.tropica, L.major and L.infantum and also exposed two genotypes (G) among the strains of L.tropica: HK-LtG1, associated with strains of L.tropica of the zymodemes MON-137 and MON-265, and HK-LtG2, associated with strains of L.tropica of the zymodemes MON-307, MON-288, MON-275 and MON-54. Following amplification and subsequent digestion by the restriction endonuclease MboI, variation in the sequence from the PGM gene also exposed two genotypes among the strains of L.tropica: PGM-G1, associated only with strains of L.tropica of the zymodeme MON-137; and PGM-G2, associated with strains of L.tropica of the zymodemes MON-265, MON-307, MON-288, MON-275 and MON-54, and, also, with six strains of L.major, five of L.infantum and one of L.donovani. The use of the HK and PGM gene sequences enabled distinction the L.tropica strains of the zymodeme MON-137 from those of the zymodeme MON-265. This genotyping system 'correctly' identified reference strains of L.tropica of known zymodemal affiliation and also from clinical samples, with a level of sensitivity down to <1 fg in the case of the former and to 1 pg of DNA in the case of the latter.
  
  
  CONCLUSIONS
  Both assays proved useful for identifying leishmanial parasites in clinical samples without resource to culture and MLEE.
authors.affiliation: Al-Quds Nutrition and Health Research Institute, Faculty of Medicine, Al-Quds University, Abu-Deis, West Bank, Palestine ; Institute of Microbiology and Hygiene, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Kifaya
authors.initials: K
authors.lastname: Azmi
authors.forename: Gabriele
authors.initials: G
authors.lastname: Schonian
authors.forename: Lionel F
authors.initials: LF
authors.lastname: Schnur
authors.forename: Abedelmajeed
authors.initials: A
authors.lastname: Nasereddin
authors.forename: Suheir
authors.initials: S
authors.lastname: Ereqat
authors.forename: Ziad
authors.initials: Z
authors.lastname: Abdeen
mesh_terms: D018912:Arabs; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D014644:Genetic Variation; D005838:Genotype; D006593:Hexokinase / Q000032:analysis / Q000235:genetics*; D006801:Humans; D007895:Leishmania tropica / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis* / Q000469:parasitology*; D018895:Microsatellite Repeats; D008877:Middle East; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D010733:Phosphoglucomutase / Q000032:analysis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA
chemical_list: D016054:DNA, Protozoan; D006593:Hexokinase; D010733:Phosphoglucomutase